Effect of bismuth subgallate (a hemostatic agent) on bone repair; a histologic, radiographic and histomorphometric study in rats.

Bismuth subgallate (BS) is a hemostatic agent used for soft tissue surgery in otorhinolaryngology and dermatology. Its effect on bone repair has not been studied. The present study undertook a quantitative and qualitative evaluation of post-extraction bone healing in the presence of BS. Under intraperitoneal anesthesia, forty male Wistar rats, 80+/-5g body weight, underwent the extraction of both lower first molars. BS was placed in the right post-extraction socket (group E) and the contralateral socket served as control (group C). The animals were killed in groups immediately, 7, 14 and 30 days post-extraction. The mandibles were resected, radiographed and processed for embedding in paraffin. The mesial socket was sectioned along the bucco-lingual axis and stained with hematoxylin-eosin. Total tissue volume and trabecular bone volume of the apical third of the sockets were determined histomorphometrically. At 14 and 30 days post-extraction, group E exhibited bone tissue that resembled that of group C. Histomorphometric analyses showed no statistically significant differences between groups C and E. Bismuth subgallate did not interfere with post-extraction bone healing. Further studies will analyze the effect of this hemostatic agent on bone repair in aniticoagulated rats.

Dynamics of recovery of morphometrical variables and pQCT-derived cortical bone properties after a short-term protein restriction in maturing rats.

Severe protein restriction during the post-weaning period in the rat markedly reduces femoral bone mass and produces a number of alterations in the shaft biomechanical properties. Body weight and femur length show an immediate and complete catch-up during nutritional rehabilitation. The aim of the present investigation was to assess whether the accelerated bone growth that occurs during protein rehabilitation is accompanied by recovery of cortical bone properties. The dynamics of the recovery of both material and geometric properties were thus evaluated on the femoral diaphyses in 45-day old female rats after a 10-day period of dietary protein restriction by peripheral quantitative computed tomography (pQCT). Protein starvation led to marked reduction of both body weight and femoral length (37% and 14% at day 10, respectively) which showed a complete catch-up after 30 d of protein refeeding. Protein restriction was associated with the interruption of the natural increase in cortical area (CtCSA), volumetric cortical bone mineral content (vCtBMC) and volumetric cortical bone mineral density (vCtBMD) which were 19.7, 25.8, and 14%, respectively, in malnourished than in control rats at the end of the protein starvation period. These parameters recovered completely during protein refeeding. Treatment also reduced by 30% both rectangular (xCSMI) and polar (pCSMI) moments of inertia. Although an improvement of these architectural indicators occurred with time, an approximately 20% deficit was still present at the end of the observation period (70 d), as was the bone strength index (BSI). It is concluded that protein restriction affected the adaptation of diaphyseal design which should reduce the mechanical competence of the femoral diaphysis because of an inadequate architectural distribution of cortical bone, and that the alteration did not show complete catch-up during the studied period.

Effects of human recombinant erythropoietin on differentiation and distribution of erythroid progenitor cells on murine medullary and splenic erythropoiesis during hypoxia and post-hypoxia.

Hemopoietic cells, the extracellular matrix, growth factors and the microenvironment are involved in the regulation of hemopoiesis. Although the regulation of erythropoiesis is well understood at the cellular level in vivo and in vitro, the role of hemopoietic sites of erythroid progenitors production has not been well defined in both steady state conditions and in stress erythropoiesis. In this study we examined the qualitative erythroid differentiation and quantitative changes of the erythroid progenitors in different erythropoietic organs during erythropoiesis of stress in a hypoxia-induced polycythemia and post-hypoxic changes in a mice model. Chronic intermittent exposure to hypobaric hypoxia induced polycythemia in mice and the post-hypoxic period was characterized by total suppression of erythropoiesis. The number and distribution in hemopoietic sites of Immature Erythroid Burst (BFU-EI), Mature Erythroid Burst (BFU-EM) and Erythroid Colony Forming Units (CFU-E) was evaluated in bone marrow and spleen of hypoxic and post-hypoxic mice after removal from the chamber. The number of BFU-EI and CFU-E, was evaluated in both femoral bone marrow and spleen of ex-hypoxic polycythemic mice, at two times intervals after the end of hypoxia. We found that in both bone marrow and spleen, the kinetics of the CFU-E pool was characterized by a sharp fall from above normal to lower than normal levels. BFU-EM increased from normal to higher than normal levels. These results have been correlated with both erythropoietin (EPO) and the erythropoietic activity. The results show that EPO levels largely control both the differentiation and the amplification of the CFU-E pool and they suggest that EPO may acts as a "survival factor" at the CFU-E level and/or increase the flow of cells from BFU-E to CFU-E. After the termination of the period of hypoxia and during post-hypoxia there was a reduction in EPO production which subsequently caused a depletion of the CFU-E population, indicating that the size of the CFU-E pool is EPO-dependent. After the injection of 1U of recombinant human erythropoietin (rHuEPO) the size of that pool was increased and the pool of BFU-EI was decreased. It is noteworthy that our studies show that the spleen functions as a large reservoir of erythroid precursors for hypoxia-induced stress erythropoiesis.

Prolonged exposure to hypobaric hypoxia transiently reduces GABA(A) receptor number in mice cerebral cortex.

The central nervous system is severely affected by hypoxic conditions, which produce alterations in neural cytoarchitecture and neurotransmission, resulting in a variety of neuropathological conditions such as convulsive states, neurobehavioral impairment and motor CNS alterations. Some of the neuropathologies observed in hypobaric hypoxia, corresponding to high altitude conditions, have been correlated with a loss of balance between excitatory and inhibitory neurotransmission, produced by alterations in glutamatergic and GABAergic receptors. In the present work, we have studied the effect of chronic hypobaric hypoxia (506 hPa, 18 h/day x 21 days) applied to adult male mice on GABA(A) receptors from cerebral cortex, to determine whether hypoxic exposure may irreversibly affect central inhibitory neurotransmission. Saturation curves for [3H]GABA specifically bound to GABA(A) receptors in isolated synaptic membranes showed a 30% decrease in maximal binding capacity after hypoxic exposure (Bmax control, 4.70+/-0.19, hypoxic, 3.33+/-0.10 pmol/mg protein), with no effect on GABA binding sites affinity (Kd control: 159.3+/-13.3 nM, hypoxic: 164.2+/-15.1 nM). Decreased B(max) values were observed up to the 10th post-hypoxic day, returning to control values by the 15th post-hypoxic day. Pharmacological properties of GABA(A) receptor were also affected by hypoxic exposure, with a 45 to 51% increase in the maximal effect by positive allosteric modulators (pentobarbital and 5alpha-pregnan-3alpha-ol-20-one). We conclude that long-term hypoxia produces a significant but reversible reduction on GABA binding to GABA(A) receptor sites in cerebral cortex, which may reflect an adaptive response to this sustained pathophysiological state.

Catch-up in mandibular growth after short-term dietary protein restriction in rats during the post-weaning period.

Catch-up growth has been defined as growth with a velocity above the statistical limits of normality for age during a defined period of time which follows a period of impaired growth. Since no data are available on catch-up in mandibular growth, the present study was designed to estimate the dynamics of the mandibular size after short-term dietary protein restriction in rats during the post-weaning period. Weanling male rats, 22 d of age, were divided into two groups, control (C) and experimental (E). E rats were fed a protein-free diet during the first 10 d; from this time on, they were placed on a 20% protein diet, as were C rats during the entire experimental period, which lasted 70 d. Five rats from both groups were randomly selected every 10 d and sacrificed. Mandibular growth was estimated directly on the right mandible by measuring several dimensions (mandibular area, base length, mandibular height, mandibular length, alveolar length and incisor alveolar process length). Alveolar and incisor alveolar process lengths did not change with age or dietary protein. All other dimensions increased with age and were thus negatively affected by protein restriction. After growth restriction ceased, the rate of increase of all affected dimensions was above normal values and deficits were swiftly eliminated. Since age-independent dimensions compose roughly the anterior portion of the mandible, this portion of the bone was not affected by protein restriction. It was, thus, the posterior part of the mandible which stopped growth during the nutritional insult and showed catch-up during nutritional rehabilitation. In summary, the rat mandible has a high potential for catch-up during the post-weaning period, showing the ability to achieve complete catch-up in about 30 d.

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells.

A simple in vivo bioassay suitable for routine testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80% suspension of heterologous (rat) red cells were used as assay animals and splenic 59Fe uptake as expression of the response to rHu-EPO. The assay took three days and the following schedule is proposed: 1) intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo bioassay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone.

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells

A simple in vivo bioassay suitable testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80 per cent suspension of heterologous (rat) red cells were used as assay animals and splenic 59 Fe uptke as expression of the response to rHu-EPO. The assay took three days and the following schedule is propose: 1)intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59 Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo biossay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells

A simple in vivo bioassay suitable testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80 per cent suspension of heterologous (rat) red cells were used as assay animals and splenic 59 Fe uptke as expression of the response to rHu-EPO. The assay took three days and the following schedule is propose: 1)intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59 Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo biossay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone(AU)

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells.

A simple in vivo bioassay suitable for routine testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80

suspension of heterologous (rat) red cells were used as assay animals and splenic 59Fe uptake as expression of the response to rHu-EPO. The assay took three days and the following schedule is proposed: 1) intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo bioassay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone.

Plasma disappearance of exogenous erythropoietin in mice under different experimental conditions.

Erythropoietin (EPO) is a glycoprotein hormone produced primarily in the kidneys and to a lesser extent in the liver that regulates red cell production. Most of the studies conducted in experimental animals to assess the role of EPO in the regulation of erythropoiesis were performed in mouse models. However, little is known about the in vivo metabolism of the hormone in this species. The present study was thus undertaken to measure the plasma tl/2 of radiolabeled recombinant human EPO (rh-EPO) in normal mice as well as in mice with altered erythrocyte production rates (EPR), plasma EPO (pEPO) titer, marrow responsiveness, red cell volume, or liver function. Adult CF-1 mice of both sexes were used throughout. For the EPO life-span studies, 30 mice in each experiment were intravenously injected with 600,000 cpm of 125l-rh-EPO and bled by cardiac puncture in groups of five every hour for 6 h. Trichloroacetic acid (TCA) was added to each plasma sample and the radioactivity in the precipitate measured in a gamma-counter. EPO, pEPO, marrow responsiveness, or red cell volume were altered by either injections of rh-EPO, 5-fluorouracil, or phenylhydrazine, or by bleeding, or red cell transfusion. Liver function was altered by CI4C administration. In the normal groups of mice, the estimated tl/2 was 182.75+/-14.4 (SEM) min. The estimated tl/2 of the other experimental groups was not significantly different from normal. These results, therefore, strongly suggest that the clearance rate of EPO in mice is not subjected to physiologic regulation and that pEPO titer can be really taken as the reflection of the EPO production rate, at least in the experimental conditions reported here.

Unexpected hypoxia-dependent erythropoietin secretion during experimental conditions not affecting tissue oxygen supply/demand ratio.

Although a great deal of evidence supports the hypothesis that plasma erythropoietin (EPO) levels of mammals are related to the oxygen supply to the tissues relative to their oxygen needs, several observation millitate against its inherent simplicity. This study presents our results obtained from in vivo experiments that suggest that hypoxia-dependent EPO production can be altered by conditions which apparently do not modify the tissue oxygen supply/demand ratio. Hypoxia-dependent EPO production rate (EPO-PR), derived from plasma EPO titers and plasma EPO half-lives, were estimated in both transfused-polycythemic and normocythemic mouse models subjected to different treatments. From calculations of the O2 carrying capacity of blood and body O2 consumption, it was assumed that the tissue supply/demand ratios were similar in both experimental and control mice of the same model at the time of induction of EPO production. The following observations were worth noting: (1) EPO-PRs in transfused polycythemic mice whose erythropoietic rates were stimulated by intermittent exposure to hypobaria (0.5 atm, 18 hr/day x 3 weeks), phenylhydrazine administration (40 mg/kg at weekly intervals x 3 weeks) or repeated rh-EPO injections (1500 U/kg 3 times a week x 3 weeks) before transfusion were more than five times high than in comparabily polycythemic mice whose erythropoietic rates were not stimulated previously; and (2) EPO-PR in response to hypobaric hypoxia was 2.08 times normal in normocythemic mice with cyclophosphamide (100 mg/kg) induced depression of erythropoiesis, and 0.33 times normal in normocythemic mice with rh-EPO (400 U/kg x 2) induced enhancement of erythropoiesis. Although the results obtained in polycythemic mice are difficult to explain, those from normocythemic mice suggest the existence of a feedback mechanism between EPO-responsive cells and EPO-producing cells. Both demonstrate the existence of experimental conditions in which modulation of the hypoxia-dependent expression of the EPO gene appears to occur. This modulation would be dependent on factors other than oxygen.

Influence of bisphosphonate on the negative erythropoietic effects of uranyl nitrate.

Uranium salts, such as uranyl nitrate, induce severe renal dysfunction and tubular necrosis and a significant impairment of both oxygen dependent erythropoietin production and response to recombinant human erythropoietin. All effects are transient and reach maximal severity on the 7th day post injection. We investigated the effects of ethane 1-hydroxy-1, 1-bisphosphonate, which counteracts the inhibitory effect of uranyl nitrate on bone formation, on the negative erythropoietic effects of uranyl nitrate. Adult female Wistar rats received 1 mg/kg body weight of uranyl acetate by the i.v. route. Ethane 1-hydroxy-1,1-bisphosphonate was injected simultaneously at a dose of 7.5 mg/kg by the same route. Seven days after drug injections, plasma erythropoietin was estimated after hypobaric hypoxemia or cobalt chloride administration. The response to exogenous erythropoietin was also measured in uranyl nitrate- and/or ethane 1-hydroxy-1,1-bisphosphonate-injected rats made polycythemic by transfusion. The erythroid response was quantitated in terms of red blood cell 59iron uptake. Ethane 1-hydroxy-1, 1-bisphosphonate counteracted the effect of uranyl nitrate on oxygen-dependent and cobalt-dependent erythropoietin production, but did not correct the right shift of the dose-response relationship for exogenous erythropoietin induced by uranyl nitrate in the polycythemic rat.

Unmodified erythropoietic response to a beta adrenergic agonist in hypothyroid mice.

beta-adrenergic agonists are able to increase erythropoiesis in the polycythemic mouse model by possibly increasing erythropoietin secretion. Since a great deal of evidence indicates that the actions of thyroid hormones and catecholamines are intimately interrelated, the present study was designed to estimate the erythropoietic response to isoproterenol, a very well-known beta-adrenergic agonist, in hypothyroid mice. Adult male CF-1 mice, maintained on a standard rodent chow and water (euthyroid) or 0.1% propylthiouracil (PTU) solution (hypothyroid) ad libitum during 37 days. Plasma T4 concentration was 1.75 +/- 0.25 micrograms/ml in euthyroid and < 1.0 microgram/ml in hypothyroid mice at this time. Mice were transfused with 1.0 ml of packed homologous red cells and the erythropoietic effect of graded doses (50, 500 and 5000 micrograms/kg) were tested by the RBC-59Fe incorporation method. No statistically significant differences (unpaired t test) were found between euthyroid and hypothyroid mice. Hypothyroidism, therefore, does not affect beta-adrenergic agonist-induced erythropoietin secretion in the present experimental conditions.

Unmodified erythropoietic response to a beta adrenergic agonist in hypothyroid mice

Los agonistas beta-adrenérgicos poseen la propriedad de estimular la eritropoyesis en el modelo del ratón policitémico mediante inducción de la secreción de eritropoyetina. Considerando la existencia de un cúmulo de evidencias que indica que las hormonas tiroideas y las catecolaminas están íntimamente relacionadas, el objetivo del presente trabajo fue estimar la potencia eritropoyética del isoproterenol, un agonista beta-adrenérgico bien conocido, en ratones hipotiroideos. Animales adultos de la cepa CF-1 fueron alimentados con dieta estandard para roedores y agua (eutiroideos) o solución al 0.1 por ciento de propiltiouracilo durante 37 dÝas. La concentración de T4 en plasma, medida por RIA, fue 1.75 mug/ml y < 1.0 mug/ml en ratones eutiroideos e hipotiroideos, respectivamente. Los animales fueron transfundidos con 1.0 ml de eritrocitos homólogos y el efecto eritropoyético de 50, 500 o 5000 pg/kg de isoproterenol estimado mediante el método de incorporación de (59)Fe al eritrón. No se observaron diferencias estadísticamente significativas (P<0.05, test de t no apareado) entre animales eutiroideos e hipotiroideos. El hipotiroidismo, por lo tanto, no afecta la secreción de eritropoyetina inducida por isoproterenol en las presentes condiciones experimentales.

Unmodified erythropoietic response to a beta adrenergic agonist in hypothyroid mice

Los agonistas beta-adrenérgicos poseen la propriedad de estimular la eritropoyesis en el modelo del ratón policitémico mediante inducción de la secreción de eritropoyetina. Considerando la existencia de un cúmulo de evidencias que indica que las hormonas tiroideas y las catecolaminas están íntimamente relacionadas, el objetivo del presente trabajo fue estimar la potencia eritropoyética del isoproterenol, un agonista beta-adrenérgico bien conocido, en ratones hipotiroideos. Animales adultos de la cepa CF-1 fueron alimentados con dieta estandard para roedores y agua (eutiroideos) o solución al 0.1 por ciento de propiltiouracilo durante 37 dYas. La concentración de T4 en plasma, medida por RIA, fue 1.75 mug/ml y < 1.0 mug/ml en ratones eutiroideos e hipotiroideos, respectivamente. Los animales fueron transfundidos con 1.0 ml de eritrocitos homólogos y el efecto eritropoyético de 50, 500 o 5000 pg/kg de isoproterenol estimado mediante el método de incorporación de (59)Fe al eritrón. No se observaron diferencias estadísticamente significativas (P<0.05, test de t no apareado) entre animales eutiroideos e hipotiroideos. El hipotiroidismo, por lo tanto, no afecta la secreción de eritropoyetina inducida por isoproterenol en las presentes condiciones experimentales. (AU)

Enhanced Erythropoietin Response to Acute Hypoxemia in Mice with Pharmacological Depression of Erythropoiesis.

The recent report of a depression of stimulated production of erythropoietin (EPO) in mice with enhanced erythropoiesis suggests that unknown mechanism (s) other than hypoxia may be involved in the regulation of EPO formation. The present study was thus designed to investigate EPO production during acute hypoxemia in a mouse model in which the oxygen-carrying capacity of blood, the plasma EPO level, and the plasma EPO half-life were within normal values in spite of a marked depression of the red cell production rate (RCPR) induced by cyclophosphamide (CP) administration. Injection of 100 mg/Kg of the drug into adult female CF-1 mice that previously received 0.4 ml of packed red cells depressed markedly the 24-hour RBC 59Fe uptake without affecting the plasma immunoreactive EPO level and the plasma disappearance of 1251-labeled recombinant human EPO. The EPO production rate, calculated from the change in plasma EPO levels and the estimated EPO clearance rate, after 4 h of exposure to hypobaric air was about 2.8 times higher in mice with CP-induced inhibition of the RCPR than in mice with normal RCPR. The results support the hypothesis that the EPO production rate in mammals is not only related to the oxygen supply to the tissues relative to their oxygen needs (main stimulus) but also to the erythroid activity of the marrow (modulatory action).

Body weight loss during acute hypoxia: effects of increased convective oxygen transport or previous acclimation.

Body weight loss and growth retardation occur in rats exposed to simulated high altitude, which may be related to the hypoxemia-induced reduction in the convective oxygen transport (COT). The present study was thus performed to determine whether transfusion polycythemia, increased affinity of hemoglobin for oxygen, or previous acclimation to hypobaria (factors that increase COT) are able to counteract its effect on body weight during the early period of exposure, which appears to be a suitable parameter to test the effectiveness of acclimatization. Polycythemia was induced in weanling rats by two ip injections of 2.5 ml/100 g b.wt of packed homologous red cells. The rise in hemoglobin O2 affinity was brought about in adult rats by giving them 0.5 g/dl sodium cyanate in the drinking water for 3 weeks. A lower body weight loss during the early period of exposure to hypobaria was seen in treated rats than in controls. However, body weight loss was still important, which would indicate that compensation was probably not complete. When growing rats were acclimated to simulated altitude, a sudden increase in body weight was observed when they were brought back to ground levels. When animals were taken to altitude again, they lost weight at a rate not significantly different to that found in non-acclimated ones. The results obtained indicate that treatments do not prevent the studied effect of hypoxia and suggest that hypophagia and the resultant initial body weight loss and secondary depression of body growth could be considered as protective mechanisms against the environmental challenge, although further investigation will be necessary to confirm the hypothesis.

Dexamethasone effects on mechanical, geometric and densitometric properties of rat femur diaphyses as described by peripheral quantitative computerized tomography and bending tests.

In previous studies with cortisol, betamethasone and oxazacort we attributed glucocorticoid effects on bone biomechanics to changes in bone mass and geometry rather than to an action on bone material properties. In this experiment, groups of 7 rats each received subcutaneous doses of 15.6, 31.2, 62.5, 125, 250, 500 or 1000 micrograms/kg per day of dexamethasone (DMS) and an additional 14 animals were controlled untreated for 4 weeks. Their fresh femurs were then scanned by peripheral quantitative computerized tomography (pQCT; XCT-960, Stratec, Germany) at the midshaft and submitted to three-point bending tests. In consonance with our earlier investigations, a significant, log-dose-related reduction in bone load-bearing capacity was observed, associated with an impairment in bone geometric properties (cross-sectional area and moment of inertia) and in body weight gain. However, the pQCT-assessed volumetric mineral density of cortical bone (vCtBMD; regarded as a material quality indicator in terms of mineralization) was significantly reduced by DMS following a dose-response relationship. Furthermore, a direct association was detected between vCtBMD and diaphyseal load-bearing capacity and stiffness. In contrast with our previous approach, data suggests that, apart from changes in bone geometric properties, glucocorticoid effects on bone material quality--as assessed by vCtBMD changes in this study--seem also to play a significant role in the determination of their biomechanical consequences.